CN112279907B - Purification method of somalupeptide - Google Patents
Purification method of somalupeptide Download PDFInfo
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Abstract
The invention discloses a method for purifying somalupeptide, which comprises the following steps: (1) Dissolving the crude product of the somalupeptide in dilute ammonia water, filtering after water bath, and collecting filtrate; (2) Taking reverse phase silica gel filler as a stationary phase, taking a salt solution with pH adjusted to 7-9 by ammonia water as a mobile phase A, taking acetonitrile as a mobile phase B, performing gradient elution, collecting fractions, and removing part of solvent by rotary evaporation to obtain a primary purification solution; (3) Taking reverse phase silica gel filler as a stationary phase, taking a salt solution with pH adjusted to 2-3 by phosphoric acid as a mobile phase A, taking a mixed solution of acetonitrile and isopropanol as a mobile phase B, performing gradient elution, collecting fractions, and removing part of solvent by rotary evaporation to obtain a secondary purification solution; (4) Taking reverse phase silica gel filler as a stationary phase, taking an aqueous solution of ammonium bicarbonate as a mobile phase A, acetonitrile as a mobile phase B, performing gradient elution, collecting fractions, performing rotary evaporation and freeze-drying to obtain the salt-free refined peptide of the somalundum with the purity of more than 99.5% and the single impurity of less than 0.1%. The method is simple and convenient to operate, and is beneficial to realizing the large-scale preparation of the somalupeptide.
Description
Technical Field
The field relates to a peptide purification method, in particular to a method for purifying the somalundin.
Technical Field
Somamunotide, named Semaglutide, is a novel long-acting glucagon-like peptide-1 (GLP-1) analog developed and produced by Daneno and Norde corporation for the treatment of type II diabetes. The somalunin has the effects of reducing blood sugar, losing weight and protecting cardiovascular system, and is approved by FDA in 10 and 18 of 2017. After the Lys side chain of the somalundum is modified by PEG, glu and octadecadicarboxylic acid, the hydrophilicity is greatly improved, and the binding force with albumin is enhanced; meanwhile, after Ala at the 2 nd position of the N end is mutated into Aib, the inactivation caused by DPP-IV enzymolysis is effectively avoided, the half life reaches 40h, patients only need to inject once a week, and the oral dosage form of the medicine is also under development at present. The CAS number of the somalundum is 910463-68-2, the molecular formula is C187H291N45O59, the molecular weight is 4113.64g/mol, and the peptide sequence is:
H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys(Octadecanedioic-γ-Glu-AEEA-AEEA)-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH。
the peptide chain of the sorulon is longer, the branched chain structure is complex, and the impurity generated in the synthesis process is more, wherein His in the sorulon 1 Is extremely easy to racemize and produce D-His 1 Impurities are very similar in physical and chemical properties to the somalundin, and thus require purification of the somalundin crude peptide. However, the prior art has poor purification effect, is insufficient to control the impurity of the simaroubde to be less than 0.1% of single impurity, and does not aim at the main impurity D-His 1 The impurities are subjected to a targeted control strategy. Therefore, a purification method of the somalundum needs to be explored to improve the purity of the somalundum, and the operation is simple and convenient, thereby being beneficial to realizing the large-scale preparation of the somalundum.
Disclosure of Invention
The invention aims at solving the technical problems of the prior art and provides a method for purifying the somalundum, which reduces single impurity to below 0.1 percent and solves the problem of the D-His of the somalundum at the same time 1 And (3) purifying impurities. In order to achieve the purpose, the purification method of the somalundin provided by the invention comprises the following steps:
step 1: dissolving the crude product of the sorulon in dilute ammonia water to obtain a crude solution of the sorulon, carrying out water bath on the crude solution in water at 45-50 ℃ for 1-2 h, filtering with 0.45 mu m, and collecting filtrate;
step 2: taking the filtrate, taking reverse phase silica gel filler as a stationary phase, taking a salt solution with pH adjusted to 7-9 by ammonia water as a mobile phase A, taking acetonitrile as a mobile phase B, carrying out gradient elution, collecting fractions, and removing part of solvent by rotary evaporation to obtain a primary purification solution of the somalupeptide;
step 3: taking a primary purification solution of the somalundum, taking reverse phase silica gel filler as a stationary phase, taking a salt solution with pH adjusted to 2-3 by phosphoric acid as a mobile phase A, taking a mixed solution of acetonitrile and isopropanol as a mobile phase B, carrying out gradient elution, collecting fractions, and removing part of solvent by rotary evaporation to obtain a secondary purification solution of the somalundum;
step 4: taking a secondary purification solution of the somalundum, taking reverse phase silica gel filler as a stationary phase, taking an aqueous solution of ammonium bicarbonate as a mobile phase A, taking acetonitrile as a mobile phase B, performing gradient elution, collecting fractions, performing rotary evaporation and freeze-drying to obtain the salt-free refined peptide of the somalundum.
Among them, the reverse phase silica gel filler is preferably an octaalkyl-bonded silica gel filler.
The octaalkyl bonding silica gel column has better selectivity on the sorulon and impurities thereof.
Preferably, the spin steaming parameters in steps 2 and 3 are as follows: the vacuum degree is less than or equal to 30 mbar at the temperature of 25-35 ℃, and the rotary steaming parameters in the step 4 are as follows: the temperature is 25-35 ℃ and the vacuum degree is less than or equal to 20 mbar.
The higher the temperature is, the higher the rotary evaporation efficiency is, the somalupeptide belongs to a polypeptide compound, and the rotary evaporation is relatively stable below 35 ℃, so that the rotary evaporation temperature is selected to be 25-35 ℃.
The concentration of the dilute ammonia water in the step 1 is preferably 1-5%, and the pH value is regulated to 7.5-9 by phosphoric acid. More preferably, the concentration of the dilute ammonia water is 2%, and the pH is adjusted to 8.0 to 8.5 by phosphoric acid.
The solubility of the somalundum is lower in an acidic condition, and the somalundum is easily dissolved in 1% -5% ammonia water, and meanwhile, the solubility of a sample is ensured and the octaalkyl bonded silica gel stationary phase is also protected under a weak alkaline condition by a crude product solution.
The salt solution in the step 2 is preferably an ammonium chloride solution of 20-100 mM/L, more preferably an ammonium chloride solution of 50mM/L, and the pH is adjusted to 8.5 by adopting ammonia water; the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 26 percent; 5-120 min: the B percent is 26 to 46 percent.
The step removes most of synthetic process impurities which are easy to remove, so that the purity of the sample reaches more than 90% after one-step purification.
The salt solution in the step 3 is preferably a phosphate solution of 20-100 mM/L, more preferably a potassium dihydrogen phosphate solution of 50mM/L, and the pH is adjusted to 2.6 by using phosphoric acid; mobile phase B was acetonitrile: isopropyl alcohol=7:3 to 9:1, and as a more preferred aspect, mobile phase B is acetonitrile: isopropanol=7:3; the elution gradient is: 0 to 5min: b percent is 10 to 45 percent; 5-120 min: the B percent is 45-70 percent.
D-His under mobile phase a ph=2.6 1 The impurity has better separation, and meanwhile, the pH=2.6 is slightly smaller than the isoelectric point of Yu Suoma lupeptide, so that the lupeptide has certain solubility and is beneficial to purification. And, in addition, the processing unit,phosphate p-somalupeptide and D-His thereof 1 The impurity has better separation effect. The addition of isopropanol into the mobile phase B can improve the separation effect, reduce the residue of the Somalin peptide on the octaalkyl bonding silica gel column, and the step can lead the purity of the Somalin peptide to be more than 99.0 percent.
In the step 4, the salt solution is preferably 10-50 mM/L ammonium bicarbonate solution, more preferably 20mM/L ammonium bicarbonate solution, and the elution gradient is as follows: 0 to 5min: the percentage of B is 10 percent to 25 percent; 5-60 min: the B percent is 25-45 percent.
After ion replacement of the cable marlutide with ammonium bicarbonate, bicarbonate ions and ammonium ions in the solution are extremely unstable, and the cable marlutide is degraded into carbon dioxide and ammonia gas under the condition of vacuum drying and then removed, so that a sample is converted into a salt-free form, and the problems of poor solubility of cable marlutide acetate water and the like are avoided. The step can ensure that the purity of the somalundum reaches more than 99.5 percent, and simultaneously controls the single impurity to be less than 0.1 percent.
Drawings
FIG. 1 is a HPLC chromatogram of a crude solution of somalupeptide of example 4
FIG. 2 is an enlarged HPLC chromatogram of a crude solution of somalupeptide of example 4
FIG. 3 is a HPLC chromatogram of one-time purification solution of somalupeptide of example 4
FIG. 4 is an enlarged HPLC chromatogram of a primary purification solution of somalupeptide of example 4
FIG. 5 is a HPLC chromatogram of a secondary purification solution of somalupeptide of example 4
FIG. 6 is an enlarged HPLC chromatogram of a secondary purification solution of somalupeptide of example 4
FIG. 7 is a HPLC chromatogram of a three-pass purified solution of somalupeptide of example 4
FIG. 8 is an enlarged HPLC chromatogram of a three-stage purification solution of somalupeptide of example 4
Detailed Description
The invention discloses a method for purifying somalupeptide. The method may be practiced by those skilled in the art with reference to the present disclosure, and it is specifically noted that all similar alterations and modifications are apparent to those skilled in the art and are considered to be included in the present invention. While the purification process of the present invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that the present invention can be modified or appropriately combined to make and use the technology of the present invention without departing from the scope of the invention.
Example 1
(1) Sample pretreatment:
2.0g of crude product of the sorulon (about 0.8g of the sorulon is contained) is taken and dissolved in 1% ammonia water solution, the pH value of the solution is regulated to 7.5 by phosphoric acid after the solution is completely dissolved, then the solution is put in a water bath at 45-50 ℃ for 1-2 h, and filtered by a 0.45um filter membrane, and the filtrate is collected as crude product of the sorulon water solution for standby.
(2) And (3) purifying in one step:
the crude water solution of the somalundin is purified by one step, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:20mM/L ammonium chloride, aqueous ammonia to ph=7, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.8g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 26 percent; 5-120 min: the B percent is 26 to 46 percent.
Collecting a sample of the fraction of the somalundum with the purity of more than 90 percent, and removing part of acetonitrile by using water bath at the temperature of 28-30 ℃ and the vacuum degree of less than 30 mbar to obtain a sample solution of the somalundum purified in one step.
(3) And (3) purifying:
the sample solution of the one-step purification of the somalupeptide is subjected to two-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:20mM/L potassium dihydrogen phosphate, aqueous solution with ph=3 adjusted with phosphoric acid, mobile phase B:70% acetonitrile +30% isopropanol; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.63g, and the elution gradient is: 0 to 5min: b percent is 10 to 45 percent; 5-120 min: the B percent is 45-70 percent.
And collecting a somalupeptide fraction sample with purity of more than 99.0% and single impurity of less than or equal to 0.2%, and removing part of acetonitrile by using water at a water bath temperature of 28-30 ℃ and a vacuum degree of less than 30 mbar to obtain a somalupeptide two-step purified sample solution.
(4) Three steps of purification:
the sample solution of the two-step purification of the somalupeptide is subjected to three-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:10mM/L ammonium bicarbonate aqueous solution, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.45g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 25 percent; 5-60 min: the B percent is 25-45 percent.
Collecting the fraction sample of the somalupeptide with purity more than 99.5% and single impurity less than or equal to 0.1%, and removing acetonitrile and most of water with water bath temperature of 28-30 ℃ and vacuum degree below 20 mbar. Obtaining the three-step purified sample solution of the somalupeptide.
(5) Freezing and drying
The three-step purification sample solution of the somalundum is frozen and dried to obtain 0.41g of the somalundum salt-free refined peptide with the purity of more than 99.5 percent and single impurity of less than 0.1 percent, and the yield is 51 percent (the HPLC spectrogram of the sample solution of each step is similar to that of the example 4).
Example 2
(1) Sample pretreatment:
2.7g of crude product of the sorulon (about containing 1.08g of the sorulon) is taken and dissolved in 5% ammonia water solution, the pH value of the solution is regulated to 9 by phosphoric acid after the crude product of the sorulon is completely dissolved, then the solution is subjected to water bath for 1-2 hours at the temperature of 45-50 ℃, and is filtered by a 0.45um filter membrane, and the filtrate is collected as crude product water solution of the sorulon for standby.
(2) And (3) purifying in one step:
the crude water solution of the somalundin is purified by one step, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:100mM/L ammonium chloride, aqueous ammonia to ph=9, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 1.08g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 26 percent; 5-120 min: the B percent is 26 to 46 percent.
Collecting a sample of the fraction of the somalundum with the purity of more than 90 percent, and removing part of acetonitrile by using water bath at the temperature of 28-30 ℃ and the vacuum degree of less than 30 mbar to obtain a sample solution of the somalundum purified in one step.
(3) And (3) purifying:
the sample solution of the one-step purification of the somalupeptide is subjected to two-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:100mM/L potassium dihydrogen phosphate, aqueous solution with ph=2 adjusted with phosphoric acid, mobile phase B:90% acetonitrile +10% isopropanol; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.85, and the elution gradient is: 0 to 5min: b percent is 10 to 45 percent; 5-120 min: the B percent is 45-70 percent.
And collecting a somalupeptide fraction sample with purity of more than 99.0% and single impurity of less than or equal to 0.2%, and removing part of acetonitrile by using water at a water bath temperature of 28-30 ℃ and a vacuum degree of less than 30 mbar to obtain a somalupeptide two-step purified sample solution.
(4) Three steps of purification:
the sample solution of the two-step purification of the somalupeptide is subjected to three-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:50mM/L ammonium bicarbonate aqueous solution, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.57g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 25 percent; 5-60 min: the B percent is 25-45 percent.
Collecting the fraction sample of the somalupeptide with purity more than 99.5% and single impurity less than or equal to 0.1%, and removing acetonitrile and most of water with water bath temperature of 28-30 ℃ and vacuum degree below 20 mbar. Obtaining the three-step purified sample solution of the somalupeptide.
(5) Freezing and drying
The three-step purification sample solution of the somalundum is frozen and dried to obtain the salt-free refined peptide of the somalundum with the purity of more than 99.5 percent and the single impurity of less than 0.1 percent, wherein the yield is 49 percent (the HPLC spectrogram of the sample solution of each step is similar to that of the example 4).
Example 3
(1) Sample pretreatment:
2.3g of crude product of the soruce peptide (about 0.92g of soruce peptide) is taken and dissolved in 2 percent ammonia water solution, after the complete dissolution, the pH value of the solution is regulated to 8 by phosphoric acid, then water bath is carried out for 1 to 2 hours at the temperature of between 45 and 50 ℃, a 0.45um filter membrane is used for filtering, and the filtrate is collected as a crude product water solution of the somalundin for standby.
(2) And (3) purifying in one step:
the crude water solution of the somalundin is purified by one step, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:50mM/L ammonium chloride, aqueous ammonia to adjust pH=8, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.92g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 26 percent; 5-120 min: the B percent is 26 to 46 percent.
Collecting a sample of the fraction of the somalundum with the purity of more than 90 percent, and removing part of acetonitrile by using water bath at the temperature of 28-30 ℃ and the vacuum degree of less than 30 mbar to obtain a sample solution of the somalundum purified in one step.
(3) And (3) purifying:
the sample solution of the one-step purification of the somalupeptide is subjected to two-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:50mM/L potassium dihydrogen phosphate, aqueous solution with pH=3 adjusted by phosphoric acid, mobile phase B:80% acetonitrile +20% isopropanol; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.73g, and the elution gradient is: 0 to 5min: b percent is 10 to 45 percent; 5-120 min: the B percent is 45-70 percent.
And collecting a somalupeptide fraction sample with purity of more than 99.0% and single impurity of less than or equal to 0.2%, and removing part of acetonitrile by using water at a water bath temperature of 28-30 ℃ and a vacuum degree of less than 30 mbar to obtain a somalupeptide two-step purified sample solution.
(4) Three steps of purification:
the sample solution of the two-step purification of the somalupeptide is subjected to three-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:30mM/L ammonium bicarbonate aqueous solution, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.53g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 25 percent; 5-60 min: the B percent is 25-45 percent.
Collecting the fraction sample of the somalupeptide with purity more than 99.5% and single impurity less than or equal to 0.1%, and removing acetonitrile and most of water with water bath temperature of 28-30 ℃ and vacuum degree below 20 mbar. Obtaining the three-step purified sample solution of the somalupeptide.
(5) Freezing and drying
The three-step purification sample solution of the somalundum is frozen and dried to obtain 0.48g of the somalundum salt-free refined peptide with the purity of more than 99.5 percent and single impurity of less than 0.1 percent, and the yield is 52 percent (the HPLC spectrogram of the sample solution of each step is similar to that of the example 4).
Example 4
(1) Sample pretreatment:
2.5g of crude product of the sorulon (about 1g of the sorulon is contained) is taken and dissolved in 2% ammonia water solution, the pH value of the solution is regulated to 8.5 by phosphoric acid after the solution is completely dissolved, then the solution is subjected to water bath for 1-2 hours at the temperature of 45-50 ℃, and is filtered by a 0.45um filter membrane, and the filtrate is collected as the crude product of the sorulon for later use. The HPLC spectrogram of the crude solution of the somalundum is shown in figure 1, the enlarged HPLC spectrogram of the crude solution of the somalundum is shown in figure 2, and the spectrogram data is shown in table 1.
TABLE 1 HPLC profile data for crude solution of somalupeptide
Detector A 215nm
| Peak# | Ret.Time | Area | Height | Area％ | Tailing Factor | Resolution | Number of Theoretical Plate |
| 1 | 7.671 | 3815 | 684 | 0.10 | -- | -- | -- |
| 2 | 7.788 | 17715 | 1781 | 0.46 | -- | -- | 6819 |
| 3 | 7.933 | 10854 | 1145 | 0.28 | -- | -- | -- |
| 4 | 8.413 | 162695 | 17777 | 4.19 | -- | -- | 16013 |
| 5 | 8.546 | 28001 | 5840 | 0.72 | -- | -- | -- |
| 6 | 9.108 | 11687 | 1133 | 0.30 | -- | -- | 14748 |
| 7 | 9.531 | 77953 | 4401 | 2.01 | -- | 1.064 | 5931 |
| 8 | 9.834 | 46825 | 4264 | 1.20 | -- | 0.709 | 11845 |
| 9 | 10.086 | 29099 | 2435 | 0.75 | -- | 0.619 | 8007 |
| 10 | 10.442 | 46422 | 3832 | 1.19 | -- | 0.878 | 13489 |
| 11 | 10.764 | 74706 | 5700 | 1.92 | -- | 0.896 | 14255 |
| 12 | 11.118 | 89354 | 6998 | 2.30 | -- | 1.012 | 17252 |
| 13 | 11.483 | 70871 | 5334 | 1.82 | -- | 0.616 | 2944 |
| 14 | 11.688 | 38155 | 5359 | 0.98 | -- | -- | -- |
| 15 | 11.934 | 2564353 | 268887 | 65.98 | 0.998 | -- | 35635 |
| 16 | 12.324 | 50815 | 3998 | 1.31 | -- | 0.737 | 3733 |
| 17 | 12.646 | 97550 | 8326 | 2.51 | -- | 0.590 | 31517 |
| 18 | 13.154 | 14999 | 1521 | 0.39 | -- | -- | -- |
| 19 | 13.366 | 97773 | 8350 | 2.52 | -- | -- | 27735 |
| 20 | 13.521 | 12735 | 2088 | 0.33 | -- | -- | -- |
| 21 | 14.138 | 16828 | 2275 | 0.43 | -- | -- | -- |
HPLC profile data for crude solution of Sodamlutide from Table 1
| Peak# | Ret.Time | Area | Height | Area％ | Tailing Factor | Resolution | Number of Theoretical Plate |
| 22 | 14.362 | 93396 | 5020 | 2.40 | -- | -- | 12348 |
| 23 | 14.692 | 3610 | 436 | 0.09 | -- | 0.350 | 1845 |
| 24 | 14.905 | 5434 | 510 | 0.14 | -- | 0.255 | 38529 |
| 25 | 15.461 | 3311 | 348 | 0.09 | 0.968 | 1.945 | 53016 |
| 26 | 16.210 | 12590 | 1197 | 0.32 | 1.114 | 2.710 | 52187 |
| 27 | 17.010 | 87787 | 7785 | 2.26 | 1.172 | 2.734 | 51106 |
| 28 | 17.494 | 23552 | 1554 | 0.61 | -- | 1.411 | 32980 |
| 29 | 17.921 | 8220 | 809 | 0.21 | -- | 1.219 | 51548 |
| 30 | 18.209 | 41497 | 4135 | 1.07 | -- | 0.679 | 18796 |
| 31 | 18.365 | 43865 | 3860 | 1.13 | -- | 0.246 | 9781 |
| Total | 3886463 | 387782 | 100.00 |
(2) And (3) purifying in one step:
the crude water solution of the somalundin is purified by one step, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:50mM/L ammonium chloride, aqueous ammonia to adjust pH=8.5, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 1g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 26 percent; 5-120 min: the B percent is 26 to 46 percent.
Collecting a sample of the fraction of the somalundum with the purity of more than 90 percent, and removing part of acetonitrile by using water bath at the temperature of 28-30 ℃ and the vacuum degree of less than 30 mbar to obtain a sample solution of the somalundum purified in one step. The HPLC spectrogram of the primary purification solution of the somalundum is shown in fig. 3, the enlarged HPLC spectrogram of the primary purification solution of the somalundum is shown in fig. 4, and the spectrogram data is shown in table 2.
TABLE 2 HPLC profile data for once purified solution of somalupeptide
Detector A 215nm
| Peak# | Ret.Time | Area | Height | Area％ | Tailing Factor | Resolution | Number of TheoreticalPlate |
| 1 | 8.389 | 1221 | 134 | 0.04 | -- | -- | 18607 |
| 2 | 9.256 | 2888 | 211 | 0.10 | -- | 2.637 | 8081 |
| 3 | 9.684 | 29317 | 2936 | 0.97 | -- | 1.276 | 22300 |
| 4 | 10.175 | 24712 | 2421 | 0.82 | -- | 1.896 | 24784 |
| 5 | 10.538 | 18295 | 1589 | 0.61 | -- | 0.693 | 2858 |
| 6 | 10.982 | 2850096 | 332316 | 94.43 | 0.932 | 0.869 | 35624 |
| 7 | 11.431 | 84155 | 9481 | 2.79 | -- | 1.903 | 36624 |
| 8 | 11.638 | 5986 | 411 | 0.20 | -- | -- | -- |
| 9 | 12.387 | 1653 | 139 | 0.05 | -- | -- | 21910 |
| Total | 3018323 | 349639 | 100.00 |
(3) And (3) purifying:
the sample solution of the one-step purification of the somalupeptide is subjected to two-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:50mM/L potassium dihydrogen phosphate, aqueous solution with pH=2.6 adjusted by phosphoric acid, mobile phase B:70% acetonitrile +30% isopropanol; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.83g, and the elution gradient is: 0 to 5min: b percent is 10 to 45 percent; 5-120 min: the B percent is 45-70 percent.
And collecting a somalupeptide fraction sample with purity of more than 99.0% and single impurity of less than or equal to 0.2%, and removing part of acetonitrile by using water at a water bath temperature of 28-30 ℃ and a vacuum degree of less than 30 mbar to obtain a somalupeptide two-step purified sample solution. The HPLC spectrogram of the two-step purification solution of the somalu peptide is shown in fig. 5, the enlarged HPLC spectrogram of the two-step purification solution of the somalu peptide is shown in fig. 6, and the spectrogram data is shown in table 3.
TABLE 3 HPLC profile data for secondary purification of somalupeptide solutions
Datector A 215nm
| Peak# | Ret.Time | Area | Height | Area％ | Tailing Factor | Resolution | Number of Theoretical Plate |
| 1 | 9.136 | 777 | 83 | 0.11 | -- | -- | 19766 |
| 2 | 9.281 | 148 | 34 | 0.02 | -- | 0.421 | 7557 |
| 3 | 9.692 | 312 | 32 | 0.04 | -- | 1.193 | 21726 |
| 4 | 10.216 | 705882 | 83809 | 99.74 | 1.036 | 2.134 | 32191 |
| 5 | 10.920 | 577 | 53 | 0.08 | -- | 1.539 | 4041 |
| Total | 707696 | 84011 | 100.00 |
(4) Three steps of purification:
the sample solution of the two-step purification of the somalupeptide is subjected to three-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:20mM/L ammonium bicarbonate aqueous solution, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.61g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 25 percent; 5-60 min: the B percent is 25-45 percent.
Collecting the fraction sample of the somalupeptide with purity more than 99.5% and single impurity less than or equal to 0.1%, and removing acetonitrile and most of water with water bath temperature of 28-30 ℃ and vacuum degree below 20 mbar. Obtaining the three-step purified sample solution of the somalupeptide. The HPLC spectrogram of the three-step purification solution of the somalu peptide is shown in fig. 7, the enlarged HPLC spectrogram of the three-step purification solution of the somalu peptide is shown in fig. 8, and the spectrogram data is shown in table 4.
TABLE 4 HPLC profile data for secondary purification of Sodamlutide
Detector A 215nm
| Peak# | Ret.Time | Area | Height | Area％ | Tailing Factor | Resolution | Number of Theoretical Plate |
| 1 | 9.161 | 436 | 52 | 0.05 | -- | -- | 20966 |
| 2 | 9.310 | 80 | 19 | 0.01 | -- | 0.357 | 4186 |
| 3 | 9.717 | 237 | 29 | 0.02 | -- | 0.997 | 25849 |
| 4 | 10.231 | 964464 | 114665 | 99.88 | 1.021 | 2.187 | 32238 |
| 5 | 10.850 | 376 | 42 | 0.04 | -- | 2.635 | 32105 |
| Total | 965594 | 114807 | 10000 |
(5) Freezing and drying
And freezing and drying the three-step purified sample solution of the somalu peptide to obtain 0.56g of the somalu peptide salt-free refined peptide with the purity of more than 99.5 percent and the single impurity of less than 0.1 percent, and the yield is 56 percent.
Example 5
(1) Sample pretreatment:
taking 3.0g of crude product of the sorulon (about 1.2g of the sorulon containing the sorulon) and dissolving the sample in 5% ammonia water solution, regulating the pH value of the solution to 9 by phosphoric acid after the sample is completely dissolved, then filtering the solution by a 0.45um filter membrane in a water bath at the temperature of 45-50 ℃ for 1-2 h, and collecting filtrate which is the crude product of the sorulon for later use.
(2) And (3) purifying in one step:
the crude water solution of the somalundin is purified by one step, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:80mM/L ammonium chloride, aqueous ammonia to ph=9, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 1.2g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 26 percent; 5-120 min: the B percent is 26 to 46 percent.
Collecting a sample of the fraction of the somalundum with the purity of more than 90 percent, and removing part of acetonitrile by using water bath at the temperature of 28-30 ℃ and the vacuum degree of less than 30 mbar to obtain a sample solution of the somalundum purified in one step.
(3) And (3) purifying:
the sample solution of the one-step purification of the somalupeptide is subjected to two-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:20mM/L potassium dihydrogen phosphate, aqueous solution with ph=3 adjusted with phosphoric acid, mobile phase B:70% acetonitrile +30% isopropanol; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.96g, and the elution gradient is: 0 to 5min: b percent is 10 to 45 percent; 5-120 min: the B percent is 45-70 percent.
And collecting a somalupeptide fraction sample with purity of more than 99.0% and single impurity of less than or equal to 0.2%, and removing part of acetonitrile by using water at a water bath temperature of 28-30 ℃ and a vacuum degree of less than 30 mbar to obtain a somalupeptide two-step purified sample solution.
(4) Three steps of purification:
the sample solution of the two-step purification of the somalupeptide is subjected to three-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:50mM/L ammonium bicarbonate aqueous solution, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.67g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 25 percent; 5-60 min: the B percent is 25-45 percent.
Collecting the fraction sample of the somalupeptide with purity more than 99.5% and single impurity less than or equal to 0.1%, and removing acetonitrile and most of water with water bath temperature of 28-30 ℃ and vacuum degree below 20 mbar. Obtaining the three-step purified sample solution of the somalupeptide.
(5) Freezing and drying
The three-step purified sample solution of the somalu peptide is frozen and dried to obtain 0.61g of the salt-free refined peptide of the somalu peptide with the purity of more than 99.5 percent and the single impurity of less than 0.1 percent, and the yield is 51 percent (the HPLC spectrogram of the sample solution of each step is similar to that of the example 4).
Example 6
(1) Sample pretreatment:
2.8g of crude product of the soruce peptide (about 1.12g of soruce peptide) is taken and dissolved in 5 percent ammonia water solution, after the complete dissolution, the pH value of the solution is regulated to 9 by phosphoric acid, then water bath is carried out for 1 to 2 hours at the temperature of between 45 and 50 ℃, a 0.45um filter membrane is used for filtering, and the filtrate is collected as a crude product water solution of the somalundin for standby.
(2) And (3) purifying in one step:
the crude water solution of the somalundin is purified by one step, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:100mM/L ammonium acetate, aqueous ammonia to adjust ph=9, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 1.12g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 26 percent; 5-120 min: the B percent is 26 to 46 percent.
Collecting a sample of the fraction of the somalundum with the purity of more than 90 percent, and removing part of acetonitrile by using water bath at the temperature of 28-30 ℃ and the vacuum degree of less than 30 mbar to obtain a sample solution of the somalundum purified in one step.
(3) And (3) purifying:
the sample solution of the one-step purification of the somalupeptide is subjected to two-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:100mM/L sodium dihydrogen phosphate, aqueous solution with ph=2 adjusted with phosphoric acid, mobile phase B:90% acetonitrile +10% isopropanol; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.87g, and the elution gradient is: 0 to 5min: b percent is 10 to 45 percent; 5-120 min: the B percent is 45-70 percent.
And collecting a somalupeptide fraction sample with purity of more than 99.0% and single impurity of less than or equal to 0.2%, and removing part of acetonitrile by using water at a water bath temperature of 28-30 ℃ and a vacuum degree of less than 30 mbar to obtain a somalupeptide two-step purified sample solution.
(4) Three steps of purification:
the sample solution of the two-step purification of the somalupeptide is subjected to three-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:50mM/L ammonium bicarbonate aqueous solution, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.57g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 25 percent; 5-60 min: the B percent is 25-45 percent.
Collecting the fraction sample of the somalupeptide with purity more than 99.5% and single impurity less than or equal to 0.1%, and removing acetonitrile and most of water with water bath temperature of 28-30 ℃ and vacuum degree below 20 mbar. Obtaining the three-step purified sample solution of the somalupeptide.
(5) Freezing and drying
The three purified sample solutions obtained were frozen and dried to obtain 0.53g of a salt-free refined peptide of somalunin having a purity of more than 99.5% and a single impurity of less than 0.1%, and the yield was 47% (the HPLC profile of the sample solution in each step was similar to that of example 4).
Example 7
(1) Sample pretreatment:
2.5g of crude product of the sorulon (about 1g of the sorulon is contained) is taken and dissolved in 2% ammonia water solution, the pH value of the solution is regulated to 8.5 by phosphoric acid after the solution is completely dissolved, then the solution is subjected to water bath for 1-2 hours at the temperature of 45-50 ℃, and is filtered by a 0.45um filter membrane, and the filtrate is collected as the crude product of the sorulon for later use.
(2) And (3) purifying in one step:
the crude water solution of the somalundin is purified by one step, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:50mM/L ammonium acetate, aqueous ammonia to adjust pH=8.5, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 1g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 26 percent; 5-120 min: the B percent is 26 to 46 percent.
Collecting a sample of the fraction of the somalundum with the purity of more than 90 percent, and removing part of acetonitrile by using water bath at the temperature of 28-30 ℃ and the vacuum degree of less than 30 mbar to obtain a sample solution of the somalundum purified in one step.
(3) And (3) purifying:
the sample solution of the one-step purification of the somalupeptide is subjected to two-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:50 mM/sodium dihydrogen phosphate, aqueous solution with ph=2.6 adjusted with phosphoric acid, mobile phase B:70% acetonitrile +30% isopropanol; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.77g, and the elution gradient is: 0 to 5min: b percent is 10 to 45 percent; 5-120 min: the B percent is 45-70 percent.
And collecting a somalupeptide fraction sample with purity of more than 99.0% and single impurity of less than or equal to 0.2%, and removing part of acetonitrile by using water at a water bath temperature of 28-30 ℃ and a vacuum degree of less than 30 mbar to obtain a somalupeptide two-step purified sample solution.
(4) Three steps of purification:
the sample solution of the two-step purification of the somalupeptide is subjected to three-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:20mM/L ammonium bicarbonate aqueous solution, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.53g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 25 percent; 5-60 min: the B percent is 25-45 percent.
Collecting the fraction sample of the somalupeptide with purity more than 99.5% and single impurity less than or equal to 0.1%, and removing acetonitrile and most of water with water bath temperature of 28-30 ℃ and vacuum degree below 20 mbar. Obtaining the three-step purified sample solution of the somalupeptide.
(5) Freezing and drying
The three-step purification sample solution of the somalundum is frozen and dried to obtain 0.48g of the somalundum salt-free refined peptide with the purity of more than 99.5 percent and single impurity of less than 0.1 percent, and the yield is 48 percent (the HPLC spectrogram of the sample solution of each step is similar to that of the example 4).
Example 8
(1) Sample pretreatment:
2.4g of crude product of the soruce peptide (about 0.96g of soruce peptide) is taken and dissolved in 4% ammonia water solution, after the complete dissolution, the pH value of the solution is regulated to 8.5 by phosphoric acid, then water bath is carried out for 1 to 2 hours at the temperature of between 45 and 50 ℃, a 0.45um filter membrane is used for filtering, and the filtrate is collected as a crude product water solution of the somalundin for standby.
(2) And (3) purifying in one step:
the crude water solution of the somalundin is purified by one step, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:70mM/L ammonium sulfate, aqueous ammonia to ph=8, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.96g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 26 percent; 5-120 min: the B percent is 26 to 46 percent.
Collecting a sample of the fraction of the somalundum with the purity of more than 90 percent, and removing part of acetonitrile by using water bath at the temperature of 28-30 ℃ and the vacuum degree of less than 30 mbar to obtain a sample solution of the somalundum purified in one step.
(3) And (3) purifying:
the sample solution of the one-step purification of the somalupeptide is subjected to two-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:85mM/L potassium dihydrogen phosphate, aqueous solution with ph=3 adjusted with phosphoric acid, mobile phase B:75% acetonitrile +25% isopropyl alcohol; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.73g, and the elution gradient is: 0 to 5min: b percent is 10 to 45 percent; 5-120 min: the B percent is 45-70 percent.
And collecting a somalupeptide fraction sample with purity of more than 99.0% and single impurity of less than or equal to 0.2%, and removing part of acetonitrile by using water at a water bath temperature of 28-30 ℃ and a vacuum degree of less than 30 mbar to obtain a somalupeptide two-step purified sample solution.
(4) Three steps of purification:
the sample solution of the two-step purification of the somalupeptide is subjected to three-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 30 x 250mm. Mobile phase a:30mM/L ammonium bicarbonate aqueous solution, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.52g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 25 percent; 5-60 min: the B percent is 25-45 percent.
Collecting the fraction sample of the somalupeptide with purity more than 99.5% and single impurity less than or equal to 0.1%, and removing acetonitrile and most of water with water bath temperature of 28-30 ℃ and vacuum degree below 20 mbar. Obtaining the three-step purified sample solution of the somalupeptide.
(5) Freezing and drying
The three purified sample solutions obtained were frozen and dried to obtain 0.47g of a salt-free refined peptide of somalunin having a purity of more than 99.5% and a single impurity of less than 0.1%, and the yield was 49% (the HPLC profile of the sample solution in each step was similar to that of example 4).
Example 9
(1) Sample pretreatment:
taking 25g of crude product of the sorulon (about 10g of sorulon containing the sorulon) and dissolving the sample in 2% ammonia water solution, regulating the pH value of the solution to 8.5 by phosphoric acid after the sample is completely dissolved, then filtering the solution in a water bath at 45-50 ℃ for 1-2 h by using a 0.45um filter membrane, and collecting filtrate which is the crude product of the sorulon for later use.
(2) And (3) purifying in one step:
the crude water solution of the somalundin is purified by one step, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 100 x 250mm. Mobile phase a:50mM/L ammonium chloride, aqueous ammonia to adjust pH=8.5, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 220ml/min, the loading amount is 10g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 26 percent; 5-120 min: the B percent is 26 to 46 percent.
Collecting a sample of the fraction of the somalundum with the purity of more than 90 percent, and removing part of acetonitrile by using water bath at the temperature of 28-30 ℃ and the vacuum degree of less than 30 mbar to obtain a sample solution of the somalundum purified in one step.
(3) And (3) purifying:
the sample solution of the one-step purification of the somalupeptide is subjected to two-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 100 x 250mm. Mobile phase a:50mM/L potassium dihydrogen phosphate, aqueous solution with pH=2.6 adjusted by phosphoric acid, mobile phase B:70% acetonitrile +30% isopropanol; the detection wavelength is 230nm, the flow rate is 220ml/min, the loading amount is 8.35g, and the elution gradient is: 0 to 5min: b percent is 10 to 45 percent; 5-120 min: the B percent is 45-70 percent.
And collecting a somalupeptide fraction sample with purity of more than 99.0% and single impurity of less than or equal to 0.2%, and removing part of acetonitrile by using water at a water bath temperature of 28-30 ℃ and a vacuum degree of less than 30 mbar to obtain a somalupeptide two-step purified sample solution.
(4) Three steps of purification:
the sample solution of the two-step purification of the somalupeptide is subjected to three-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 100 x 250mm. Mobile phase a:20mM/L ammonium bicarbonate aqueous solution, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 220ml/min, the loading amount is 6.12g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 25 percent; 5-60 min: the B percent is 25-45 percent.
Collecting the fraction sample of the somalupeptide with purity more than 99.5% and single impurity less than or equal to 0.1%, and removing acetonitrile and most of water with water bath temperature of 28-30 ℃ and vacuum degree below 20 mbar. Obtaining the three-step purified sample solution of the somalupeptide.
(5) Freezing and drying
The three-step purification sample solution of the somalu peptide is frozen and dried to obtain 5.71g of the salt-free refined peptide of the somalu peptide with the purity of more than 99.5 percent and the single impurity of less than 0.1 percent, and the yield is 57 percent (the HPLC spectrogram of the sample solution of each step is similar to that of the example 4).
Example 10
(1) Sample pretreatment:
taking 50g of crude product of the sorulon (about 20g of sorulon containing the sorulon) and dissolving the sample in 2% ammonia water solution, regulating the pH value of the solution to 8.5 by phosphoric acid after the sample is completely dissolved, then filtering the solution in a water bath at 45-50 ℃ for 1-2 h by using a 0.45um filter membrane, and collecting filtrate which is the crude product of the sorulon for later use.
(2) And (3) purifying in one step:
the crude water solution of the somalundin is purified by one step, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 150 x 250mm. Mobile phase a:50mM/L ammonium chloride, aqueous ammonia to adjust pH=8.5, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 400ml/min, the loading amount is 20g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 26 percent; 5-120 min: the B percent is 26 to 46 percent.
Collecting a sample of the fraction of the somalundum with the purity of more than 90 percent, and removing part of acetonitrile by using water bath at the temperature of 28-30 ℃ and the vacuum degree of less than 30 mbar to obtain a sample solution of the somalundum purified in one step.
(3) And (3) purifying:
the sample solution of the one-step purification of the somalupeptide is subjected to two-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 150 x 250mm. Mobile phase a:50mM/L potassium dihydrogen phosphate, aqueous solution with pH=2.6 adjusted by phosphoric acid, mobile phase B:70% acetonitrile +30% isopropanol; the detection wavelength is 230nm, the flow rate is 400ml/min, the loading amount is 16.21g, and the elution gradient is: 0 to 5min: b percent is 10 to 45 percent; 5-120 min: the B percent is 45-70 percent.
And collecting a somalupeptide fraction sample with purity of more than 99.0% and single impurity of less than or equal to 0.2%, and removing part of acetonitrile by using water at a water bath temperature of 28-30 ℃ and a vacuum degree of less than 30 mbar to obtain a somalupeptide two-step purified sample solution.
(4) Three steps of purification:
the sample solution of the two-step purification of the somalupeptide is subjected to three-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octaalkyl bonded silica gel column, chromatographic column specification is 150 x 250mm. Mobile phase a:20mM/L ammonium bicarbonate aqueous solution, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 400ml/min, the loading amount is 11.46g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 25 percent; 5-60 min: the B percent is 25-45 percent.
Collecting the fraction sample of the somalupeptide with purity more than 99.5% and single impurity less than or equal to 0.1%, and removing acetonitrile and most of water with water bath temperature of 28-30 ℃ and vacuum degree below 20 mbar. Obtaining the three-step purified sample solution of the somalupeptide.
(5) Freezing and drying
The three-step purification sample solution of the somalu peptide is frozen and dried to obtain 10.79g of the salt-free refined peptide of the somalu peptide with the purity of more than 99.5 percent and the single impurity of less than 0.1 percent, and the yield is 54 percent (the HPLC spectrogram of the sample solution of each step is similar to that of the example 4).
Comparative example
(1) Sample pretreatment:
2.5g of crude product of the sorulon (about containing 1g of the sorulon) is taken and dissolved in 60% acetonitrile water solution, and then the mixture is subjected to water bath for 1 to 2 hours at the temperature of between 45 and 50 ℃ and is filtered by a 0.45um filter membrane, and the filtrate is collected as the crude product of the sorulon water solution for later use.
(2) And (3) purifying in one step:
the crude water solution of the somalundin is purified by one step, and chromatographic conditions are as follows: preparing a chromatographic column: octadecyl bonded silica gel column, column size was 30 x 250mm. Mobile phase a:50mM/L sodium bicarbonate, aqueous ammonia to adjust pH=6.5, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 1g, and the elution gradient is: 0 to 5min: the percentage of B is 10 percent to 26 percent; 5-120 min: the B percent is 26 to 46 percent.
Collecting a sample of the fraction of the somalundum with the purity of more than 90 percent, and removing part of acetonitrile by using water bath at the temperature of 28-30 ℃ and the vacuum degree of less than 30 mbar to obtain a sample solution of the somalundum purified in one step.
(3) And (3) purifying:
the sample solution of the one-step purification of the somalupeptide is subjected to two-step purification, and chromatographic conditions are as follows: preparing a chromatographic column: octadecyl bonded silica gel column, column size was 30 x 250mm. Mobile phase a:50mM/L potassium sulfate, aqueous phosphate pH=3, mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, the loading amount is 0.55g, and the elution gradient is: 0 to 5min: b percent is 10 to 45 percent; 5-120 min: the B percent is 45-70 percent.
And collecting a somalundum fraction sample with purity more than 95% and single impurity less than or equal to 0.4%, and removing part of acetonitrile by using water bath at the temperature of 28-30 ℃ and the vacuum degree of less than 30 mbar to obtain a somalundum two-step purification sample solution.
(4) Salt conversion:
performing salt conversion on the cable-marlutide two-step purified sample solution, and performing chromatographic conditions: preparing a chromatographic column: octadecyl bonded silica gel column, column size was 30 x 250mm. Mobile phase a: water; mobile phase B: acetonitrile; the detection wavelength is 230nm, the flow rate is 25ml/min, and the sample loading amount is 0.31g; first, washing with a 50mM/L ammonium acetate solution (pH=7.0) containing 5% acetonitrile for 15min; finally, gradient elution is carried out, wherein the elution gradient is as follows: 0 to 5min: the percentage of B is 10 percent to 25 percent; 5-60 min: the B percent is 25-45 percent.
Collecting a sample of the fraction of the somalupeptide with the purity of more than 98.5 percent and the single impurity of less than or equal to 0.2 percent, and removing acetonitrile and most of water by using a water bath at the temperature of 28-30 ℃ and the vacuum degree of less than 20 mbar. Obtaining the sample solution of the somalupeptide salt.
(5) Freezing and drying
And freezing and drying the obtained three purified sample solutions to obtain 0.25g of the refined peptide of the somalunin acetate with the purity of more than 98.5 percent and the single impurity of more than 0.1 percent and the yield of 25 percent.
Claims (15)
1. A method for purifying somalundin, comprising the steps of:
step 1: dissolving the crude product of the somalundum in dilute ammonia water to obtain a solution of the crude product of the somalundum, filtering after water bath, and collecting filtrate;
step 2: taking the filtrate, taking a reversed phase octaalkyl bonded silica gel filler as a stationary phase, taking an ammonium chloride salt solution with pH value adjusted to 7-9 and with concentration of 20-100 mM/L by ammonia water as a mobile phase A, taking acetonitrile as a mobile phase B, carrying out gradient elution, collecting fractions with purity of more than 90%, and removing part of solvent by rotary evaporation to obtain a primary purification solution of the somalundum;
step 3: taking a primary purification solution of the somalundum, taking a reversed phase octaalkyl bonding silica gel filler as a stationary phase, taking a potassium dihydrogen phosphate salt solution with the pH adjusted to 2.6 and 20-100 mM/L by phosphoric acid as a mobile phase A, taking a mixed solution of acetonitrile and isopropanol as a mobile phase B, carrying out gradient elution, collecting fractions with the purity of more than 99.0% and the single impurity of less than or equal to 0.2%, and removing part of solvent by rotary evaporation to obtain a secondary purification solution of the somalundum;
step 4: taking a secondary purification solution of the somalundum, taking a reversed phase octaalkyl bonding silica gel filler as a stationary phase, taking an aqueous solution of 10-50 mM/L ammonium bicarbonate as a mobile phase A, taking acetonitrile as a mobile phase B, carrying out gradient elution, collecting fractions with purity of more than 99.5% and single impurity of less than or equal to 0.1%, carrying out rotary evaporation and freeze-drying to obtain the salt-free refined peptide of the somalundum.
2. The method for purifying the somalundin according to claim 1, wherein: the rotary steaming parameters in the steps 2 and 3 are as follows: the temperature is 25-35 ℃ and the vacuum degree is less than or equal to 30 mbar.
3. The method for purifying the somalundin according to claim 1, wherein: the rotary steaming parameters in the step 4 are as follows: the temperature is 25-35 ℃ and the vacuum degree is less than or equal to 20 mbar.
4. The method for purifying the somalundin according to claim 1, wherein: the concentration of the dilute ammonia water in the step 1 is 1-5%, and the pH value is regulated to 7.5-9 by phosphoric acid.
5. The method for purifying the somalundin according to claim 1, wherein: the concentration of the dilute ammonia water in the step 1 is 2%, and the pH value is regulated to 8.0-8.5 by phosphoric acid.
6. The method for purifying the somalundin according to claim 1, wherein: the water bath condition in the step 1 is as follows: the temperature is as follows: 45-50 ℃ for 1-2 h.
7. The method for purifying the somalundin according to claim 1, wherein: the pH of the ammonium chloride salt solution in the step 2 is 8.5.
8. The method for purifying the somalundin according to claim 1, wherein: the concentration of the ammonium chloride salt solution in the step 2 is 50mM/L.
9. The method for purifying the somalundin according to claim 1, wherein: the gradient in the step 2 is as follows: 0 to 5min: the percentage of B is 10 percent to 26 percent; 5-120 min: the B percent is 26 to 46 percent.
10. The method for purifying the somalundin according to claim 1, wherein: the concentration of the potassium dihydrogen phosphate solution in the step 3 is 50mM/L.
11. The method for purifying the somalundin according to claim 1, wherein: the mobile phase B in the step 3 is acetonitrile: isopropanol=7:3 to 9:1.
12. The method for purifying the somalundin according to claim 1, wherein: the mobile phase B in the step 3 is acetonitrile: isopropanol=7:3.
13. The method for purifying the somalundin according to claim 1, wherein: the gradient in the step 3 is as follows: 0 to 5min: b percent is 10 to 45 percent; 5-120 min: the B percent is 45-70 percent.
14. The method for purifying the somalundin according to claim 1, wherein: the concentration of the aqueous solution of ammonium bicarbonate in the step 4 is 20mM/L.
15. The method for purifying the somalundin according to claim 1, wherein: the gradient in the step 4 is as follows: 0 to 5min: the percentage of B is 10 percent to 25 percent; 5-60 min: the B percent is 25-45 percent.
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| CN201910685603.2A CN112279907B (en) | 2019-07-27 | 2019-07-27 | Purification method of somalupeptide |
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